牛分枝杆菌Mb1230基因的表达纯化及其活性评价

doi:10.16431/j.cnki.1671-7236.2015.10.005

›› 2015, Vol. 42 ›› Issue (10): 2544-2550.doi: 10.16431/j.cnki.1671-7236.2015.10.005

牛分枝杆菌Mb1230基因的表达纯化及其活性评价

李明¹, 贾红¹, 鑫婷¹, 郭晓宇¹, 袁维锋¹, 侯邵华¹, 侯强¹, 高新桃¹, 吴竞¹, 董瑞凯¹, 杨宏军², 刘来兴², 朱鸿飞¹

1. 中国农业科学院北京畜牧兽医研究所, 北京 100193;
2. 山东省农业科学院奶牛研究中心, 济南 250183

收稿日期:2015-01-21 出版日期:2015-10-20 发布日期:2015-10-23
通讯作者: 朱鸿飞 E-mail:bioclub@vip.sina.com
作者简介:李明(1987-),男,云南丽江人,硕士,研究方向:疫苗制备与免疫学,E-mail:liming112117@163.com
基金资助:
中国农业科学院北京畜牧兽医研究所中央级公益性科研院所基本科研业务费专项资金项目(2014ywf-zd-1)

Expression,Purification and Activity Evaluation of Mb1230 Gene of Mycobacterium bovis

LI Ming¹, JIA Hong¹, XIN Ting¹, GUO Xiao-yu¹, YUAN Wei-feng¹, HOU Shao-hua¹, HOU Qiang¹, GAO Xin-tao¹, WU Jing¹, DONG Rui-kai¹, YANG Hong-jun², LIU Lai-xing², ZHU Hong-fei¹

1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2. Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan 250183, China

Received:2015-01-21 Online:2015-10-20 Published:2015-10-23

摘要/Abstract

摘要： 为了探究牛分枝杆菌Mb1230蛋白在牛结核病诊断中的应用价值,本试验利用PCR方法扩增出Mb1230基因,构建重组质粒pET-22b-Mb1230,经IPTG诱导表达后用亲和层析纯化重组蛋白,通过结核菌素皮内变态反应试验(TST试验)、IFN-γ释放试验(IGRA试验)和间接ELISA对重组蛋白的活性进行评价。SDS-PAGE和Western blotting结果显示,重组蛋白大小与理论值相符,且能与鼠抗His的抗体反应有特异性条带;在TST试验、IGRA试验和间接ELISA中也表现出抗原活性。结果表明,重组Mb1230蛋白具有良好的B细胞活性和T细胞活性,在牛结核病诊断中具有应用潜力。

关键词: 牛分枝杆菌; Mb1230; 原核表达; 牛结核病诊断

Abstract: To explore the value of Mb1230 protein of Mycobacterium bovis in the diagnosis of bovine tuberculosis,we obtained the Mb1230 gene by PCR and constructed recombinant plasmid pET-22b-Mb1230.Recombinant Mb1230 protein was obtained by IPTG induction and purified by affinity chromatography.The activity of the recombinant protein was evaluated by TST test,IGRA test and indirect ELISA.The size of the recombinant protein matched with the theoretical value proved by SDS-PAGE;Western blotting result showed that the recombinant protein could react with mouse anti-His antibody,and had specific band;The results of TST test,IGRA test and indirect ELISA test also showed the recombinant protein had antigenic activity.The results indicated the recombinant protein Mb1230 had good B cell and T cell activity,so,it had the potential application in the diagnosis of bovine tuberculosis.

Key words: Mycobacterium bovis; Mb1230; prokaryotic expression; diagnosis of bovine tuberculosis

中图分类号:

S852.61⁺8

李明, 贾红, 鑫婷, 郭晓宇, 袁维锋, 侯邵华, 侯强, 高新桃, 吴竞, 董瑞凯, 杨宏军, 刘来兴, 朱鸿飞. 牛分枝杆菌Mb1230基因的表达纯化及其活性评价[J]. , 2015, 42(10): 2544-2550.

LI Ming, JIA Hong, XIN Ting, GUO Xiao-yu, YUAN Wei-feng, HOU Shao-hua, HOU Qiang, GAO Xin-tao, WU Jing, DONG Rui-kai, YANG Hong-jun, LIU Lai-xing, ZHU Hong-fei. Expression,Purification and Activity Evaluation of Mb1230 Gene of Mycobacterium bovis[J]. , 2015, 42(10): 2544-2550.

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牛分枝杆菌Mb1230基因的表达纯化及其活性评价

Expression,Purification and Activity Evaluation of Mb1230 Gene of Mycobacterium bovis

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